Real-time visualization of mRNA synthesis during memory formation in live mice

Significance Arc is one of the genes that are rapidly transcribed by neuronal activity and thus used as a marker for memory trace or engram cells. However, the dynamics of engram cell populations is not well-known because of the difficulty in monitoring the rapid and transient gene expression in live animals. Using a mouse model in which endogenous Arc messenger RNA (mRNA) is fluorescently labeled, we demonstrate that Arc-expressing neuronal populations have distinct dynamics in different brain regions and that only a small subpopulation that consistently expresses Arc during both memory encoding and retrieval exhibits context-specific calcium activity. This live-animal RNA-imaging technique will offer a powerful tool for connecting gene expression to neuronal activity patterns and to behavior.

Immunofluorescence 12-13-week-old male mice were housed in the home cage and habituated by handling for 3 days. Then, the mice were subject to CFC and returned to the home cage for 1. With a vibratome (Leica, VT1000s), brains were sectioned coronally to 50 μ m, permeabilized with 1% Triton X (Thermo Fisher Scientific, 28314) in Tris-buffered saline (TBS) for 30 min at room temperature (RT), and incubated in 0.4% Triton X and 5% normal goat serum (NGS) (Vector Labs, S-1000) in TBS for 30 min at RT. Sections were treated with primary antibodies in 0.4% Triton X and 5% NGS in TBS at 4 °C overnight and then washed three times with 0.1% Triton X in TBS for 20 min at RT. After secondary antibody treatment for 3 hr at RT, the sections were counterstained with 1 μg/ml DAPI (Thermo Fisher Scientific, 62248) in 0.1% Triton X in TBS. Then, the sections were washed with 0.1% Triton X in TBS for 20 min at RT and then twice with TBS for 20 min, and cover-slipped with VectaShield mounting media (Vector Labs, H-1700). Anti-Arc (1:1000, Synaptic Systems, 156 003) was used as primary antibody, and goat-anti rabbit IgG conjugated to Alexa Fluor 555 (A21428; 1:200) was used as secondary antibody.

Simulations
To compare the accuracy of GERI and conventional IEG promoter-based reporter expression methods for identifying IEG-positive neurons, we generated random Arc activation traces for 500 neurons. At the time of stimulation, 25% of the neurons were activated during 4 min to mimic the CFC experiment. The basal level of IEG activation was varied from 0-0.5% per min. The IEG activation traces were then convolved with the response function of GERI or that of shGFP reporter (1). The response function of GERI was obtained from the average intensity profile of the Arc transcription sites (n = 42) over time. The response function of shGFP was constructed from data in a previous report (2).
The fraction of IEG-positive neurons was calculated by counting the number of neurons in which the intensity exceeded a threshold of 30% of the peak intensity at 6 min after stimulation for GERI and at 1 hr after stimulation for shGFP, respectively. The simulation was repeated 10 times. The accuracy was calculated by dividing the number of correctly predicted neurons by the number of total neurons (n = 500).

Electrical stimulation of cultured hippocampal neurons
Electrical burst stimulation was applied to cultured hippocampal neurons through two thin  To image jRGECO1a in fixed brain tissues, we used a wide-field fluorescence microscope

Place cell identification
Place cells were defined using a method similar to an approach described previously (3).
The 3-m-long virtual track was first divided into 150 position bins (2 cm per bin). Using the ΔF/F traces obtained during running (speed >1.5 cm/s and run length > 5 cm), we calculated the sum of ΔF/F for each bin and normalized by the dwell time in each bin to generate a ΔF/F field map. The resulting ΔF/F field map was smoothed by computing the moving average with a sliding window of three bins. The potential place field was identified as a region exceeding the median value of the ΔF/F field map. Place cells were then identified using the following criteria: 1) the potential field is wider than 20 cm; 2) the mean value of the ΔF/F field map in the potential field must be 2.2 times higher than the mean value of the ΔF/F field map outside the potential field; and 3) Ca 2+ events must be present in at least 15% of the visits to the potential field. To evaluate whether each mouse actually distinguished virtual Contexts A and B, we calculated spatial correlation.
For neurons identified as place cells on day 1, we calculated the Pearson's correlation coefficient of the ΔF/F field map between the same or different contexts. The spatial information was calculated using the following formula (5): where λ is the Ca 2+ event rate, λi is the mean Ca 2+ event rate in the i th place bin, and Pi is 8 the probability that the mouse stays in the i th place bin.

Network graph
We first converted the Ca 2+ event traces into binary and temporally binned (1 sec per bin) traces. The resulting traces were used to generate a correlation matrix by calculating Pearson's correlation coefficients. We then generated a binary adjacent matrix with pairs that were statistically significant (p < 0.05) and had a correlation coefficient higher than 0.1. The network graph was visualized using Gephi (https://gephi.org/) software with the ForceAtlas2 layout. We calculated degrees, cluster coefficients, and modularity using custom written MATLAB scripts. The degree kv denotes the number of edges in each neuron. The normalized degree was calculated by dividing kv by (the number of neurons in network -1). Clustering coefficients were calculated using the following formula (6): where A is the adjacent matrix in which Aij is a binary value indicating whether the i th neuron and the j th neuron are correlated. The normalized modularity was calculated using the following formula (7): where m is the number of edges in the network, ti denotes the module (Arc++/non-Arc++ or place cell/non-place cell) of the i th neuron, and δ is a delta function.    burst rate, inferred theta-burst rate, and mean spike number per burst of each group were plotted (* P < 0.05, ** P < 0.01, *** P < 10 -10 by rank-sum test). Generally, neurons in the  contexts. (E) Comparison of A1-Arc-and A1-Arc+ neurons in terms of their spatial correlation between place fields when mice were exposed to the same (A-A) contexts.